Foot-and-mouth disease is a highly contagious disease of great economic importance, afflicting primarily cloven-hoofed animals. The mortality directly attributable to foot-and-mouth disease is comparatively low, generally, but in young animals the mortality can be quite high. Of greater economic importance, the disease is so debilitating that infected animals cannot economically be raised and fed. The only recognized effective procedure for eliminating the infection once it has been discovered is to destroy all infected animals, disinfect all premises which have been occupied by the animals, and decompose the carcasses in quicklime. Since the infection spreads extremely rapidly, the economic foundation of entire communities or regions can be destroyed by one major outbreak of foot-and-mouth disease.
Vaccines have been produced which immunize against foot-and-mouth disease, primarily, by inactivation or attenuation of virus. Such vaccines have been found to be effective in some measure, but outbreaks of foot-and-mouth disease have been linked to vaccines in which the virus was incompletely inactivated or insufficiently attenuated. Infections have also been traced to the escape of virus from facilities devoted to research on foot-and-mouth disease or to production of foot-and-mouth disease vaccines.
Foot-and-mouth disease (FMD) is caused by a picornavirus of the family Aphthovirus. There are several viral serotypes of foot-and-mouth virus (FMDV), the most common of which are identified by the serotype designations A, O and C, and less common identified as SAT-1, SAT-2, SAT-3 and ASIA-1.
FMDV has been described in some detail; see, for example, (H. L. Backrach, in Beltsville Symposium on Agricultural Research, J. A. Romberger, Ed. (Allanheld, Montclair, N.J. 1977), pp. 3-32; Annual Reviews of Microbiology, 22, 201 (1968).) The molecular biology of these viruses have been described, R. R. Rueckert, in Molecular Biology of Picornavizuses, R. Perez-Bercoff, Ed. (Plenum, New York, 1979), p. 113. The virus has a molecular size of about 7.times.10.sup.6 daltons and contains a plus-stranded RNA genome of approximately 8,000 nucleotides. Picornavirus proteins have been synthesized in infected cells as a precursor of a protein that is subsequently processed by cellular and virus-coded proteases into four major capsid proteins (VP.sub.1, VP.sub.2, VP.sub.3, and VP.sub.4) and numerous non-capsid proteins. The VP.sub.3 protein when used to inoculate swine ellicited a neutralizing anti-body response and protected both swine and cattle from infection. (J. Laporte, et al., C.R. Acad. Sci. 276, 3399 (1973); H. L. Backrach, et al., J. Immunol. 115, 1636 (1975); H. L. Backrach, et al., Vet. Microbiol.) Based upon this information, Dennis G. Kleid, et al. Science, Vol. 214, 4 Dec. 1981, 1125-1129, were able to produce a cloned viral protein vaccine for foot-and-mouth disease which gave anitbody responses in cattle and swine. Recombinant DNA molecules and processes for producing peptides with the specificity of foot-and-mouth disease viral antigens is described in UK patent application GB No. 2,079,288A, 20 Jan. 1982. K. Strohmaier, et al., Proc. 5th Int. Congress Virology, Strasbourg, 1981, have suggested that regions of the VP protein sequence between positions 146 and 155 and positions 200-213 would be effective in inducing antibodies to the foot-and-mouth disease virus; however, no affirmative demonstration of the use of these regions as antigens has been reported. This VP.sub.1 sequence corresponds to the VP.sub.3 sequence described earlier in the United States; see explanation by Meloen, A. H., J.Gen.Virol. (1979) 45, 761-763.
In the past antigens have been obtained in several fashions, including derivation from natural materials, coupling of a hapten to a carrier, and by recombinant DNA technology. Sela, et al. (Proc. Nat. Acad. Sci., U.S.A., Vol. 68, No. 7, pp. 1450-1455, July 1971; Science, Vol. 166, pp. 1365-1374, December 1969; Adv. Immun., Vol. 5, pp. 29-129, 1966) have also described certain synthetic antigens.
Antigens derived from natural materials are the countless number of known antigens which occur naturally, such as blood group antigens, HLA antigens, differentiation antigens, viral and bacterial antigens, and the like. Considerable effort has been expended over the last century in identifying and studying these antigens.
Certain "synthetic" antigens have been prepared by coupling small molecules to carriers such as, for example, bovine serum albumin, thus producing antigens which will cause production of antibody to the coupled small molecule. The carrier molecule is necessary because the small molecule itself would not be "recognized" by the immune system of the animal into which it was injected. This technique has also been employed in isolated instances to prepare antigens by coupling peptide fragments of known proteins to carriers, as described in the above-referenced Sela et al. articles.
While this hapten-carrier technique has served the research community well in its investigations of the nature of the immune response, it has not been of significant use to produce antigens which would play a role in diagnostic or therapeutic modalities. The reasons for this deficiency are several.
First, to choose and construct a useful antigenic determinant from a pathogen by this technique, one must determine the entire protein sequence of the pathogen to have a reasonable change of success. Because of the difficulty of this task it has rarely, if ever, been done.
Classically, vaccines are manufactured by introducing killed or attenuated organisms into the host along with suitable adjuvents to initiate the normal immune response to the organisms while, desirably, avoiding the pathogenic effects of the organism in the host. The approach suffers from the well known limitations in that it is rarely possible to avoid the pathogenic response because of the complexity of the vaccine which includes not only the antigenic determinant of interest but many related and unrelated deleterious materials, any number of which may, in some or all individuals, induce an undesirable reaction in the host. For example, vaccines produced in the classical way may include competing antigens which are detrimental to the desired immune response, antigens which include unrelated immune responses, nucleic acids from the organism or culture, endotoxins and constituents of unknown composition and source. These vaccines, generated from complex materials, inherently have a relatively high probability of inducing competing responses even from the antigen of interest.
Recombinant DNA technology has opened new approaches to vaccine technology which does have the advantage that the manufacture begins with a monospecific gene; however, much of this advantage is lost in actual production of antigen in E. coli, or other organisms. In this procedure, the gene material is introduced into a plasmid which is then introduced into E. coli which produces the desired protein, along with other products of the metabolism, all in mixture with the nutrient. This approach is complicated by the uncertainty whether the desired protein will be expressed in the transformed E. coli. Further, even though the desired protein may be produced, there is uncertainty as to whether or not it can be harvested or whether it will be destroyed in the process of E. coli growth. It is well known, for example, that foreign or altered proteins are digested by E. coli. Even if the protein is present in sufficient quantities to be of interest, it must still be separated from all of the other products of the E. coli metabolism, including such deleterious substances as undesired proteins, endotoxins, nucleic acids, genes and unknown or unpredictable substances. Finally, even if it were possible, or becomes possible through advanced, though necessarily very expensive, techniques, to separate the desired protein from all other products of the E. coli metabolism, the vaccine still comprises an entire protein which may include undesirable antigenic determinants, some of which are known to initiate very serious responses. Indeed, it is known that certain proteins which could otherwise be considered as vaccines include an antigenic determinant which induce such serious cross reference or side reactions as to prevent the use of the material as a vaccine.
It is also possible, using hybridoma technology, to produce antibodies to viral gene products. Basically, hybridoma technology allows one to begin with a complex mixture of antigens and to produce monospecific antibodies later in the process.
In contrast, the present invention is the reverse process, in that we start with the ultimate in high purity antigenic determinant and thus avoid the necessity for purification of the desired antigenic product.
Hybridoma antibodies are known to be of low avidity and low binding constant, and therefore, have limited value.
Ultimately, in hybridoma technology, one must rely on the production of the antibody by cells which are malignant, with all of the attendant concerns regarding separation techniques, purity and safety.
Hybridoma production relies upon tissue culture or introduction into mice, with the obvious result that production is costly and there is inherent variability from lot to lot.
In addition, it is difficult to make a hybrid to molecules which comprise only a small percentage of the complex mixture one must start with.
Previous studies by Arnon et al. (1971, Proc. Nat. Acad. Sci. 68:1450), Atassi (1975, Immunochemistry 12:423) and Vyas et al. (1972 Science 178:1300) have been interpreted by these authors to indicate that short linear amino acid sequences are, in general, unlikely to elicit antibodies reactive with the native protein structure. It was thought that for most regions of most molecules, antigenic determinants resulted from amino acid residues well separated in the linear sequence but conformation of the peptides used to elicit antibodies was thought to be critical in most cases, even for those antigens involving amino acids close together in a sequence. Lerner, et al., Cell 23:109-110, 1981; Nature 287:801-805 (1980), discovered that antibodies to linear peptides react with native molecules. Elaborate biosyntheses thus became unnecessary, uneconomical and obsolete.
Notwithstanding the availability of inactivated or attenuated virus vaccines against foot-and-mouth disease, there has remained a very great economic and practical demand, and great theoretical interest in the development of a vaccine against foot-and-mouth disease which would be free of the risks which have heretofore attended the manufacture and handling of the FMDV which causes the disease. The availability of cloned viral proteins may well be a very significant step forward from the older and very risky approaches. But the cloned viral protein vaccine approach also carries with it a number of inherent disadvantages, limitations and risks. Variations in the biosynthesis system itself may cause variation in expression of proteins, thus affecting purity, yields, potency, etc. of antigens. The possibility of finding live or active organisms in the resulting product is always a potential risk. in addition, the presence of other proteins, and difficult and inefficient separations, suggest the likelihood that vaccines produced through the cloned viral protein route will not be monospecific. Indeed, Kleid, et al. suggested that there was evidence that the antibodies to VP.sub.3 elicit antibodies to several different antigenic sites. Thus, purity, potency, and safety are major concerns with products derived from this technology.
Notwithstanding that the general concept of preparing synthetic antigens, starting either from a known peptide sequence or from a genome have been described, and notwithstanding that the synthesis of peptides of suitable length for use in antigenic materials is now quite well known, there remains a very large area of antigen-antibody technology which continues to defy predictability. While there are some guidelines and some suggestions as to possible antigenic sequences, the field remains largely a matter of speculation, and of trial and error. Even with the recognition that a long sequence may contain antigenically active constituents, there remains a great deal of uncertainty and speculation as to whether all or only part of the sequence is required for antigenicity, and whether or not a smaller portion of the sequence would be of greater or lesser antigenicity.